Insulin-stimulated phosphorylation of protein phosphatase 1 regulatory subunit 12B revealed by HPLC-ESI-MS/MS
- Equal contributors
1 Center for Metabolic and Vascular Biology, Arizona State University, Tempe, AZ, USA
2 Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy/Health Sciences, Wayne State University, 259 Mack Ave., Detroit, MI, USA
Proteome Science 2012, 10:52 doi:10.1186/1477-5956-10-52Published: 1 September 2012
Protein phosphatase 1 (PP1) is one of the major phosphatases responsible for protein dephosphorylation in eukaryotes. Protein phosphatase 1 regulatory subunit 12B (PPP1R12B), one of the regulatory subunits of PP1, can bind to PP1cδ, one of the catalytic subunits of PP1, and modulate the specificity and activity of PP1cδ against its substrates. Phosphorylation of PPP1R12B on threonine 646 by Rho kinase inhibits the activity of the PP1c-PPP1R12B complex. However, it is not currently known whether PPP1R12B phosphorylation at threonine 646 and other sites is regulated by insulin. We set out to identify phosphorylation sites in PPP1R12B and to quantify the effect of insulin on PPP1R12B phosphorylation by using high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry.
14 PPP1R12B phosphorylation sites were identified, 7 of which were previously unreported. Potential kinases were predicted for these sites. Furthermore, relative quantification of PPP1R12B phosphorylation sites for basal and insulin-treated samples was obtained by using peak area-based label-free mass spectrometry of fragment ions. The results indicate that insulin stimulates the phosphorylation of PPP1R12B significantly at serine 29 (3.02 ± 0.94 fold), serine 504 (11.67 ± 3.33 fold), and serine 645/threonine 646 (2.34 ± 0.58 fold).
PPP1R12B was identified as a phosphatase subunit that undergoes insulin-stimulated phosphorylation, suggesting that PPP1R12B might play a role in insulin signaling. This study also identified novel targets for future investigation of the regulation of PPP1R12B not only in insulin signaling in cell models, animal models, and in humans, but also in other signaling pathways.